Preparation Of Monoclonal Antibodies Against Vibrio Cholerae O1 Serotype Ogawa And Identification Of Epitope Mimic Peptide Of The Monoclonal Antibodies

Cholera is an acute dehydrating diarrheal disease caused by vibrio cholerae. Cholera, which occurs rapidly and spreads widely, is classified as an international quarantine infectious disease and the first type infectious disease in China. Based on recordation, it has caused worldwide epidemic for seven times, and epidemics as well as pandemics of cholera are a major health problem for human. Vibrio cholerae strains are subclassified over 200 strains, however, only these belonging to the O1 and O139 serogroups are capable of causing epidemic and pandemic cholera. V. cholerae O1 can be classified as classical or E1 Tor biotypes, serotypes Ogawa and Inaba, while there is no further classification of V. cholerae O139. Each serotype produces the similar cholera toxin, causing the similar clinic symptoms.The response to Cholera epidemic is well-organized exigent responsive form,which can prevent the death caused by Cholera, however, can not prevent Cholera occurrence. There is always a specially dominating serotype in Cholera epidemic and the predominant strain changes frequently. Thus, it is important to set up a rapid responsive mechanism and strategy. The establishment of rapid detection of Cholera will be helpful to monitor and prevent Cholera epidemic. The monoclonal antibodies (McAbs) against Vibrio cholerae conjugating to colloidal gold can detect Vibrio cholerae rapidly, simply, exactly, and its application does not need special apparatus, which is suitable for usage on scene and the elementary medical institution. Vaccine is the most effective means to extinguish Cholera epidemic, and the long-lasting combination of medium-term and long-term prevention measure is important and emphasized. Thus, the development of safe and effective new vaccine is necessary and the identification of effective vaccine epitope candidates is the key. The phage display random library proves to be a high-through screening means for the antigen epitope selection.In the study, we used killed Vibrio cholerae O1 Serotype Ogawa, Inaba and Vibrio cholerae O139 to prepare McAb against Vibrio cholerae by the Hybridoma cell lines. We got two strains of McAbs with high specificity and activity, IXiao3G6 McAb and IXiao1D9 McAb, by one step lateral flow test. Then the McAbs were further developed to the Monoclonal antibodies conjugating to colloidal gold against Vibrio cholerae, which provides rapidly, simply and exactly detecting means for diagnosis of clinic cholera cases, inspection of highly dangerous human, investigation of epidemiology and sanitation quarantine. Based on that, the purified IXiao3G6 McAb was used to biospan in phage random 7 peptide library. The mimic epitopes were acquired after 3 rounds of biospanning. The recombinant vectors containing the mimic epitopes repeating in series and the gene encoding cholerae toxin subunit B (CTB) were constructed and expressed in prokaryotic expression E. coli BL21. And the identification of its antigenicity would be helpful for the development of eiptope mimic peptide vaccine against Vibrio cholerae and be important for the establishment of effective and rapid responsive mechanism and strategy.PART IPREPARATION AND SCREENING OF MONOCLONAL ANTIBODIES AGAINST VIBRIO CHOLERAEObjectives: To prepare and screen the monoclonal antibodies against Vibrio Cholerae and to provide the effective antibodies for prevetion and diagnosis of Cholera.Methods: Balb/c mice were immunized with attenuated Vibrio cholerae O1 Serotype Ogawa, Inaba and Vibrio cholerae O139, respectively. Hybridoma cell lines secreting McAbs against Vibrio cholerae were established. The specificity and crossreactivity of McAb was identified by indirect enzyme-linked immunosorbent assay (ELISA) as the first biospanning. The biospanning used the thalli antigen of Vibrio cholerae O1 Serotype Ogawa, Inaba, Vibrio cholerae O139, other Vibrio strains and other ordinary bacteria. The second biospanning used the"the monoclonal antibodies conjugating to colloidal gold parallel screening"to set up the monoclonal antibody cell lines assembly screening methods, which combined the monoclonal antibodies conjugating to colloidal gold and the pyroxylin membranes coated with the purified antibodies.Results: Total of 602 McAb-positive hybridoma cell lines against Vibrio cholerae O1 Serotype Ogawa, 386 against Vibrio cholerae O1 Serotype Inaba and 82 against Vibrio cholerae O139 were identified by indirect ELISA. Then, by repetitive indirect ELISA and across responsive screening, 15 cell strains specifically secreting McAb against Vibrio cholerae O1 Serotype Ogawa, 15 specifically against Vibrio cholerae O1 Serotype Inaba and 10 specifically against Vibrio cholerae O139 were obtained. After assembly screening, only two lines against cholerae O1 Serotype Ogawa with high specificity and activity, X8 (IXiao3G6)和X2 (IXiao1D9), were obtained.Conclusions: Two specific McAbs against Vibrio cholerae O1 Serotype Ogawa were obtained successfully, which could be very helpful for the early diagnosis and further research of Vibrio cholerae O1 Serotype Ogawa.PART IIIDENTIFICATION AND APPLICATION OF SPECIFIC MONOCLONAL ANTIBODIES AGAINST VIBRIO CHOLERAE O1 SEROTYPE OGAWAObjectives: To identify the physical-chemical and biological properties of the two specific cell lines against cholerae O1 Serotype Ogawa, X8 (IXiao3G6)和X2 (IXiao1D9), and then develop the monoclonal antibody conjugating to colloidal gold against cholerae O1 Serotype Ogawa, to establish the rapid, super sensitive detection method of"double-antibody-sandwich"membrane colloidal gold based on monoclonal antibodies, which was used for the early diagnosis of Cholera.Methods: To separate, purify and identify the two highly specific strains of monoclonal antibodies, and further purify with Protein A affinity Chromatography. Then analyze the chromosome of the McAbs, identify the subtypes, measure the titer and the affinity constant, and analyze its specificity. To research the antigen epitope identifying by the two strains of McAbs by combined experimental site, indirect ELISA with LPS of cholerae O1 Serotype Ogawa and Western blot assays. To prepare the colloidal gold-antibody conjugates and responsive membranes. To develop the monoclonal antibody conjugating to colloidal gold against cholerae and to estimate its specificity, sensitivity, repeatability and stability.Results: SDS-PAGE showed the purity of purified monoclonal antibodies was over 95%. The subtypes of the two strains of monoclonal antibodies were identified as IgG2b and IgG3, respectively, which had high specificity, high affinity (affinity constant was 109), high titer (ascites titer was 10-6). The two strains of monoclonal antibodies were targeted to different antigen epitope: IXiao3G6 was targeted to the LPS of cholerae O1 Serotype Ogawa, while IXiao1D9 was targeted to the non-LPS sites of cholerae O1 Serotype Ogawa. The rapid detection monoclonal antibodies conjugating to colloidal gold against cholerae O1 Serotype Ogawa was developed to specifically detect the thalli antigen of cholerae O1 Serotype Ogawa and establish the detection method of monoclonal antibodies sandwich-colloidal gold for Vibrio Cholerae. The results demonstrated that this method had high specificity(100%), sensitivity(limit of detection is 1×104cfu/ml), repeatability(100%), and stability(1 year at room temperature), and could detect rapidly(5min) and simply.Conclusions: The separation, purification and identification of the two highly specific strains of monoclonal antibodies further demonstrated that they could be used as the diagnosis reagent, which would be useful for further screening the eiptope mimic peptides to obtain the mimic peptide which could induce protecting immune response. The colloidal gold against cholerae O1 Serotype Ogawa could detect Vibrio Cholerae, which served as an effective detection measure for clinic diagnosis, food safety detection, environment sanitation supervision, counter-terrorism activity and immigration quarantine. Through our researching work, we tackled the trouble of distinguishing the cholerae O1 Serotype Ogawa and Inaba, which was a blank in the area of detection of Cholera.PART IIISCREENING AND IDENTIFICATION OF EIPTOPE MIMIC PEPTIDE OF THE MONOCLONAL ANTIBODIES AGAINST VIBRIO CHOLERAE O1 SEROTYPE OGAWAObjectives: To obtain the mimic peptide highly binding to cholerae O1 Serotype Ogawa by using purified IXiao3G6 McAb to biospan in phage random 7 peptide library, and to establish the basement for further research of the mimic peptide vaccine of Vibrio cholerae. Methods: The purified IXiao3G6 McAb was used to biospan in phage random 7 peptide library and mimic epitopes were acquired after 3 rounds of biospanning. The"double-antibody-sandwich"ELISA and the specific inhibition test were used to analyze the combining ability and specificity of the obtained phage short peptide and the IXiao3G6 McAb. The DNA sequence analysis was used to analyze the sequence of the positive clones and the related bioinformatic tools was used to analyze the alignments.Results: After three rounds of biospanning, the positive phages were enriched effectively. 22 phage clones were positive of the 25 phage clones selected randomly by"double-antibody-sandwich"ELISA. The DNA sequence analysis showed that the amino acids sequence of 20 positive clones were identical completely, which was identified as APAIPAS. Alignment analysis showed that no homologous known sequence. The specific inhibition test showed that the LPS of cholerae O1 Serotype Ogawa could inhibit the binding of the positive clones with IXiao3G6 McAb, and the inhibitory intensity was enhanced by increment of the content of LPS, while the LPS of control bacteria did not show the corresponding inhibitory effect. These results further demonstrated phage display peptide could be mimetic to the antigen epitope of the LPS of cholerae O1 Serotype Ogawa.Conclusions: The mimic peptide mimesis to the antigen epitope of the LPS of cholerae O1 Serotype Ogawa served as the basement for development of the mimic peptide vaccine of Vibrio cholerae.PART IVRECOMBINANT EXPRESSION AND ANTIGENICITY ANALYSIS OF THE MIMIC PEPTIDE OF CHOLERAE O1 SEROTYPE OGAWA AND CHOLERAE TOXIN BObjectives: To construct recombinant vectors containing genes encoding cholerae toxin B(CTB) and serial repeating mimic peptide to the LPS of cholerae O1 Serotype Ogawa, express the recombinant plasmids in prokaryotic expression E. coli BL21, and then identify the antigenicity. To establish the basement for further research and clinic application of mimic peptide vaccine of Vibrio cholerae.Methods: The target genes encoding CTB were amplified by PCR. After digested with Kpn I and BamH I simultanously, the purified PCR products were inserted into the compatible sites of prokaryotic expression vectors pET32a (+), by using T4 DNA ligase at a molar ratio of 4:1 at 4℃overnight, then the ligation products were transformed to E. coli JM109. The recombinant vectors were analyzed by DNA sequence analysis to identify the recombinant vector pET32a(+)-CTB. To construct the recombinant plasmid pET32a(+)-CTB-MP by inserting the DNA fragments of the mimic peptide of the LPS of cholerae O1 Serotype Ogawa with mimic peptide(MP) gene repeated in series downstream to the recombinant plasmid pET32a(+)-CTB. The recombinant vectors were transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. The target proteins were purified by Ni-NTA reagent and the antigenicity was identified by indirect ELISA, Western-Blot assay and the specific inhibition test.Results: The recombinant plasmids with serial repeated MP of LPS of cholerae O1 Serotype Ogawa and CTB were constructed. SDS-PAGE showed CTB-MP and CTB proteins both highly expressed by IPTG induction, with relative molecular weight of 34KD and 32KD, respectively, as expected. Both were purified via Ni-NTA with 90% purity. Indirect ELISA and Western-Blot assay showed CTB-MP fusing proteins could bind to the monoclonal antibodies against IXiao3G6. The specific inhibition test demonstrated that CTB-MP could inhibit combination of the monoclonal antibodies against IXiao3G6 and LPS of cholerae O1 Serotype Ogawa. CTB could interact with GM-1.Conclusions: The MP of LPS of cholerae O1 Serotype Ogawa could interact with monoclonal antibodies against IXiao3G6 and inhibit the combination of the monoclonal antibodies against IXiao3G6 and LPS of cholerae O1 Serotype Ogawa, which suggested the MP would be mimetic to the epitopes of LPS of cholerae O1 Serotype Ogawa. CTB could interact with GM-1, which suggested the CTB has a respond in the fusion MP protein.